CN111072752B - Preparation method of polypeptide capable of promoting human skin cell proliferation, product and application thereof - Google Patents

Preparation method of polypeptide capable of promoting human skin cell proliferation, product and application thereof Download PDF

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CN111072752B
CN111072752B CN201911364980.2A CN201911364980A CN111072752B CN 111072752 B CN111072752 B CN 111072752B CN 201911364980 A CN201911364980 A CN 201911364980A CN 111072752 B CN111072752 B CN 111072752B
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CN111072752A (en
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孙培冬
王培宇
杨成
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Jiangnan University
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Abstract

The invention discloses a preparation method of polypeptide for promoting human skin cell proliferation, a product and application thereof, wherein the pumpkin seed polypeptide prepared by ultrafiltration is passed through sephadex G-10 and is freeze-dried to obtain polypeptide powder; preparing 1-50 mg/mL solution of polypeptide separated and prepared by sephadex G-10, adjusting pH to 5.0-10.0, and sequentially passing through 0.45 mu m and 0.22 mu m microporous filter membranes to obtain screened pumpkin seed polypeptide; injecting the swelled DEAE-52 anion exchange resin into a chromatographic column, balancing 2 column volumes by deionized water, carrying out gradient elution on a sample at the flow rate of 1mL/min, collecting eluent of 50 th to 100 th tubes with 3mL of eluent per tube, and carrying out freeze drying to obtain the polypeptide capable of promoting human skin cell proliferation and identify the polypeptide sequence, wherein the polypeptide sequence is shown as SEQ ID No. 1. The polypeptide compound extracted by the invention can obviously promote the proliferation of human skin fibroblasts and human immortalized keratinocytes, can be used as a repairing agent and an anti-aging component to be added into skin care products, and has wide market prospect.

Description

Preparation method of polypeptide capable of promoting human skin cell proliferation, product and application thereof
Technical Field
The invention belongs to the technical field of biology and new medicines, and particularly relates to a preparation method of polypeptide capable of promoting human skin cell proliferation, a product and application thereof.
Background
Cells are the largest organs in the human body, and human aging is to some extent cell aging. In the aging process of skin, with the aging of keratinocytes and fibroblasts, epidermis becomes thinner and inferior, dermis becomes collagen and elastin are reduced, and skin is wrinkled and thickened and has loss of elasticity. One of the important manifestations of cellular senescence is therefore the loss of the proliferative capacity of the cell.
The pumpkin is a trailing herbaceous plant of Cucurbitaceae Cucurbita, is native to south America, and is wide in planting range and rich in resources. Semen Cucurbitae contains polysaccharides, amino acids, vitamins and other nutrients, and adenine and trigonelline and other alkaloids.
After the pumpkin seeds are treated, the oil and fat in the pumpkin seeds can be extracted by adopting a squeezing method, a leaching method, a supercritical fluid extraction method and the like, the protein content of the pumpkin seed meal after oil extraction can reach more than 50 percent, and the pumpkin seed meal is a high-quality plant protein resource. However, at present, the pumpkin seed meal is only used as feed or is discarded, so that not only is the resource waste caused, but also the environmental pollution is caused.
Research shows that the pumpkin seed protein not only has the functions of reducing blood sugar and resisting oxidation, but also has the functions of inhibiting proliferation, inducing apoptosis and inducing differentiation on various tumor cells. At present, researches on pumpkin seed polypeptides and preparation processes thereof are focused on the aspects of oxidation resistance and the like, and no report is provided on researches on the aspect of promoting cell proliferation of pumpkin seed polypeptides.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made keeping in mind the above and/or other problems occurring in the prior art.
Therefore, the present invention aims at providing the preparation process of polypeptide capable of promoting human skin cell proliferation.
In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of polypeptide capable of promoting human skin cell proliferation comprises preparing 1-50 mg/mL solution of pumpkin seed polypeptide, adjusting pH to 5.0-10.0, centrifuging, collecting supernatant, sequentially passing through 0.45 μm and 0.22 μm microporous filter membranes, adjusting ultrafiltration pressure to 0.1-0.2 MPa, ultrafiltering at room temperature for 1-10 times, collecting polypeptide solution passing through 1000Da ultrafiltration membrane, and freeze drying to obtain polypeptide powder; adding the pumpkin seed polypeptide prepared by ultrafiltration into sephadex G-10, eluting with ultrapure water as an eluent after the sample completely permeates into the sephadex, collecting the eluent, and freeze-drying to obtain polypeptide powder; preparing 1-50 mg/mL solution of polypeptide separated and prepared by sephadex G-10, adjusting pH to 5.0-10.0, centrifuging, collecting supernatant, and sequentially passing through 0.45-micrometer and 0.22-micrometer microporous filter membranes to obtain screened pumpkin seed polypeptide; injecting the swelled DEAE-52 anion exchange resin into a chromatographic column, balancing 2 column volumes by deionized water, adding the screened pumpkin seed polypeptide, performing gradient elution on the sample by using 0, 0.5 and 1mol/L NaCl solutions at the flow rate of 1mL/min respectively, eluting 3mL in each tube, collecting the eluates of 50 th to 100 th tubes, and performing freeze drying to obtain the polypeptide capable of promoting human skin cell proliferation.
As a preferred embodiment of the preparation method of the polypeptide with the function of promoting human skin cell proliferation, the method comprises the following steps: the polypeptide capable of promoting human skin cell proliferation is obtained by freeze drying, wherein the temperature of a cold trap is-50 to-45 ℃, the vacuum degree is 0.025MPa, and the drying time is 24 h.
As a preferred embodiment of the preparation method of the polypeptide with the function of promoting human skin cell proliferation, the method comprises the following steps: adding the pumpkin seed polypeptide prepared by ultrafiltration into sephadex G-10, and eluting by using ultrapure water as an eluent after the sample completely permeates into the gel, wherein the elution condition is that the elution speed is 1.0mL/min, and collecting one tube of eluent every 5 min.
As a preferred embodiment of the preparation method of the polypeptide with the function of promoting human skin cell proliferation, the method comprises the following steps: and eluting by using ultrapure water as an eluent, and collecting the eluent, namely collecting the eluent of the 20 th to 30 th pipes.
As a preferred embodiment of the preparation method of the polypeptide with the function of promoting human skin cell proliferation, the method comprises the following steps: preparing 1-50 mg/mL solution of pumpkin seed polypeptide, adjusting the pH value to 5.0-10.0, centrifuging and collecting supernatant, wherein the centrifugation condition is 8000r/min, and the time is 10 min.
As a preferred embodiment of the preparation method of the polypeptide with the function of promoting human skin cell proliferation, the method comprises the following steps: the preparation method of the pumpkin seed polypeptide comprises the steps of adding desalted pumpkin seed protein into deionized water according to the material-liquid ratio of 1: 5-1: 40g/mL, carrying out ultrasonic treatment for 10-60 min at the power of 20-60 Hz to obtain a pumpkin seed protein dispersion liquid, adjusting the temperature to 30-60 and the pH to 5.0-10.0, adding alkaline protease according to the enzyme bottom ratio of 1-5.5%, carrying out enzymolysis for 2-4 h, then carrying out inactivation, adjusting the temperature of a reaction liquid to 25-50 ℃ and the pH to 7.5, adding trypsin according to the enzyme bottom ratio of 1-5.5%, carrying out enzymolysis for 2-4 h, then carrying out inactivation, cooling the mixed liquid to the room temperature, adding 1mol/L HCl to adjust the pH to 7.0, centrifuging for 25min at the speed of 5000r/min, collecting a supernatant, and carrying out freeze drying to obtain the pumpkin seed polypeptide.
As a preferred embodiment of the preparation method of the polypeptide with the function of promoting human skin cell proliferation, the method comprises the following steps: the preparation method of the desalted pumpkin seed protein comprises the steps of adding degreased pumpkin seeds into deionized water according to a material-liquid ratio of 1: 10-1: 50g/mL, adjusting the pH value to 7.0-11.0, performing ultrasonic extraction at 25-50 ℃ for 240min, wherein the ultrasonic power is 20Hz in the first 120min, the ultrasonic power is 40Hz in the second 120min, centrifuging to collect supernatant, adjusting the pH value to 7.0, centrifuging at 5000r/min for 25min, collecting precipitate, washing with water to remove salt, and performing freeze drying to obtain the desalted pumpkin seed protein.
As a preferred embodiment of the preparation method of the polypeptide with the function of promoting human skin cell proliferation, the method comprises the following steps: the preparation method of the degreased pumpkin seeds comprises the steps of crushing the pumpkin seeds by a high-speed crusher, sieving the crushed pumpkin seeds by a 100-mesh sieve, adding petroleum ether according to the material-liquid ratio of 1: 2-1: 5g/mL, stirring for 4 hours at the temperature of 25-50 ℃, carrying out suction filtration, repeating for three times, removing residual petroleum ether in a fume hood after degreasing, and carrying out low-temperature sealed storage.
Another purpose of the invention is to provide a polypeptide prepared by the preparation method of the polypeptide for promoting human skin cell proliferation, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.
The invention also aims to provide application of the polypeptide prepared by the preparation method of the polypeptide for promoting human skin cell proliferation as an anti-aging, repairing, protecting and antioxidant component in cosmetics.
The invention has the beneficial effects that:
(1) the invention provides a preparation method of polypeptide capable of promoting human skin cell proliferation, wherein a polypeptide compound is extracted from pumpkin seeds for the first time, the extracted polypeptide compound can obviously promote the proliferation of human skin fibroblasts and human immortalized keratinocytes, can be used as a repairing agent and an anti-aging component to be added into skin care products, and has wide market prospect.
(2) The invention provides a preparation method of polypeptide capable of promoting human skin cell proliferation, wherein in the preparation process of pumpkin seed polypeptide, desalted pumpkin seed protein is used as a raw material, preferably, the pumpkin seed polypeptide is prepared by alkaline-trypsin double-enzyme composite enzymolysis, the polypeptide yield is higher, the polypeptide with the molecular weight less than 1000Da reaches 92.72%, the antioxidation performance is optimal, the cell proliferation promoting performance is better, and the activities of HSF and HaCat cells respectively reach 116.15% and 121.02%. Combining the pumpkin seed polypeptide prepared by ultrafiltration, preparing a 1-50 mg/mL solution by using the polypeptide prepared by separation of sephadex G-10, adjusting the pH to 5.0-10.0, centrifuging, collecting supernatant, and sequentially passing through 0.45 mu m and 0.22 mu m microporous filter membranes to obtain the screened pumpkin seed polypeptide; injecting the swelled DEAE-52 anion exchange resin into a chromatographic column, eluting the sample by 0, 0.5 and 1mol/L NaCl solution at the flow rate of 1mL/min in a gradient manner, preferably collecting the eluent of 50 th to 100 th tubes with 3mL of eluent per tube, and freeze-drying to obtain the polypeptide capable of promoting the proliferation of the skin cells of the human body, wherein the synergistic effect of the steps is realized, and the cell proliferation performance is optimal.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a graph showing the elution profile of the polypeptides separated by Sephadex G-10 in examples 1-3 of the present invention.
FIG. 2 is a graph showing the effect of G-10-isolated fraction on cell proliferation in examples 1 to 3 of the present invention.
FIG. 3 is a graph showing the elution profile after DEAE-52 separation in examples 4 to 6 of the present invention.
FIG. 4 is a graph showing the effect of DEAE-52 fractions on cell proliferation in examples 4 to 6 of the present invention.
FIG. 5 is a graph showing the separation result of DEAE-52 in comparative example 1 of the present invention.
FIG. 6 is a graph showing the separation result of DEAE-52 in comparative example 2 of the present invention.
FIG. 7 is a graph showing the degree of proteolysis of pumpkin seeds in comparative example 3 of the present invention.
FIG. 8 is a graph showing the yield of pumpkin seed polypeptides in comparative example 3 of the present invention.
FIG. 9 is a graph showing the antioxidant activity of the polypeptide of comparative example 3 of the present invention.
FIG. 10 is a graph showing the effect of the polypeptide of comparative example 3 of the present invention on cell proliferation.
FIG. 11 is an analytical HPLC chromatogram of component F2-b in example 6 of the present invention.
FIG. 12 is a UV absorption spectrum of component F2-b in example 6 of the present invention.
FIG. 13 is a low energy 1: TOF-MS spectrum of component F2-b in example 6 of the present invention.
FIG. 14 is a high energy 2 TOF-MS spectrum of component F2-b in example 6 of the present invention.
FIG. 15 shows the structural identification of F2-b in example 6 of the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The polypeptide of the invention can promote the human skin cell proliferation activity determination:
the proliferation activity of pumpkin seed polypeptides on Human Skin Fibroblasts (HSF) and human immortalized keratinocytes (HaCat) was examined. The cell culture medium containing only DMEM as a control group was subjected to measurement of the absorbance OD at 490nm after 24 hours, and the cell viability was calculated according to the formula (1).
Figure BDA0002338178340000051
In the formula: ODSample (I)The absorbance value of the sample group is obtained; ODBlank spaceBlank absorbance values; ODControlThe absorbance of the control group is shown.
Example 1
(1) Preparation of defatted pumpkin seeds
Crushing the pumpkin seeds by a high-speed crusher, and sieving by a 60-mesh sieve. Adding petroleum ether according to a feed-liquid ratio of 1:4(g/mL), stirring at 40 ℃ for 4h, performing suction filtration, repeating for 3 times, degreasing, removing residual petroleum ether in a fume hood, and storing at low temperature in a sealed manner.
(2) Preparation of pumpkin seed protein
Adding defatted semen Cucurbitae into deionized water at a ratio of 1:30g/mL, adjusting pH to 9.5, extracting at 35 deg.C for 180min, centrifuging, collecting supernatant, and adjusting pH to 4.3. Centrifuging, collecting precipitate, washing with water to neutrality, and freeze drying to obtain semen Cucurbitae protein.
(3) Preparation method of pumpkin seed polypeptide
Adjusting the temperature and pH of the pumpkin seed protein dispersion to 50 deg.C and 9.0, adding alkaline protease at enzyme bottom ratio of 2.5%, performing enzymolysis for 2 hr, and inactivating in boiling water. Adjusting the temperature and pH of the reaction solution to 37 deg.C and 7.5, adding trypsin, performing enzymolysis for 2 hr, and inactivating. During the enzymolysis, 0.5mol/L NaOH solution is added dropwise to maintain the pH value of the solution unchanged. And after the enzymolysis is finished, cooling the mixed solution to room temperature, adding 1mol/L HCl to adjust the pH value to an isoelectric point, centrifuging to collect supernatant, and freeze-drying to obtain the polypeptide.
(4) Ultrafiltration
Preparing 10mg/mL solution of pumpkin seed polypeptide, sequentially passing through 0.45-micrometer and 0.22-micrometer microporous filter membranes, adjusting ultrafiltration pressure to 0.1-0.2 MPa, and ultrafiltering at room temperature for 5 times. Collecting the polypeptide solution passing through 1000Da ultrafiltration membrane, and freeze-drying to obtain polypeptide.
(5) Sephadex G-10 isolation
Adding the pumpkin seed polypeptide with the mass concentration of 40mg/mL into the sephadex, restoring the liquid level to a position 5cm away from the gel by using ultrapure water after the sample completely permeates into the gel, and eluting by using the ultrapure water as an eluent. The elution conditions were: the elution rate was 1.0 mL/min. Collecting one tube of eluent every 5min, collecting 20-30 tubes of eluent, measuring the absorbance at 274nm, and drawing an elution curve, which is shown in figure 1 (named as F2).
Example 2
(1) Preparation of defatted pumpkin seeds
Crushing the pumpkin seeds by a high-speed crusher, and sieving by a 60-mesh sieve. Adding petroleum ether according to a feed-liquid ratio of 1:4(g/mL), stirring at 40 ℃ for 4h, performing suction filtration, repeating for 3 times, degreasing, removing residual petroleum ether in a fume hood, and storing at low temperature in a sealed manner.
(2) Preparation of pumpkin seed protein
Adding defatted semen Cucurbitae into deionized water at a ratio of 1:30g/mL, adjusting pH to 9.5, extracting at 35 deg.C for 180min, centrifuging, collecting supernatant, and adjusting pH to 4.3. Centrifuging, collecting precipitate, washing with water to neutrality, and freeze drying to obtain semen Cucurbitae protein.
(3) Preparation method of pumpkin seed polypeptide
Adjusting the temperature and pH of the pumpkin seed protein dispersion to 50 deg.C and 9.0, adding alkaline protease at enzyme bottom ratio of 2.5%, performing enzymolysis for 2 hr, and inactivating in boiling water. Adjusting the temperature and pH of the reaction solution to 37 deg.C and 7.5, adding trypsin, performing enzymolysis for 2 hr, and inactivating. During the enzymolysis, 0.5mol/L NaOH solution is added dropwise to maintain the pH value of the solution unchanged. And after the enzymolysis is finished, cooling the mixed solution to room temperature, adding 1mol/L HCl to adjust the pH value to an isoelectric point, centrifuging to collect supernatant, and freeze-drying to obtain the polypeptide.
(4) Ultrafiltration
Preparing 10mg/mL solution of pumpkin seed polypeptide, sequentially passing through 0.45-micrometer and 0.22-micrometer microporous filter membranes, adjusting ultrafiltration pressure to 0.1-0.2 MPa, and ultrafiltering at room temperature for 5 times. Collecting the polypeptide solution passing through 1000Da ultrafiltration membrane, and freeze-drying to obtain polypeptide.
(5) Sephadex G-10 isolation
Adding the pumpkin seed polypeptide with the mass concentration of 40mg/mL into the sephadex, restoring the liquid level to a position 5cm away from the gel by using ultrapure water after the sample completely permeates into the gel, and eluting by using the ultrapure water as an eluent. The elution conditions were: the elution rate was 1.0 mL/min. Collecting one tube of eluent every 5min, collecting 1-19 tubes of eluent, measuring the absorbance at 274nm, and drawing an elution curve, which is shown in figure 1 (named as F1).
Example 3
(1) Preparation of defatted pumpkin seeds
Crushing the pumpkin seeds by a high-speed crusher, and sieving by a 60-mesh sieve. Adding petroleum ether according to a feed-liquid ratio of 1:4(g/mL), stirring at 40 ℃ for 4h, performing suction filtration, repeating for 3 times, degreasing, removing residual petroleum ether in a fume hood, and storing at low temperature in a sealed manner.
(2) Preparation of pumpkin seed protein
Adding defatted semen Cucurbitae into deionized water at a ratio of 1:30g/mL, adjusting pH to 9.5, extracting at 35 deg.C for 180min, centrifuging, collecting supernatant, and adjusting pH to 4.3. Centrifuging, collecting precipitate, washing with water to neutrality, and freeze drying to obtain semen Cucurbitae protein.
(3) Preparation method of pumpkin seed polypeptide
Adjusting the temperature and pH of the pumpkin seed protein dispersion to 50 deg.C and 9.0, adding alkaline protease at enzyme bottom ratio of 2.5%, performing enzymolysis for 2 hr, and inactivating in boiling water. Adjusting the temperature and pH of the reaction solution to 37 deg.C and 7.5, adding trypsin, performing enzymolysis for 2 hr, and inactivating. During the enzymolysis, 0.5mol/L NaOH solution is added dropwise to maintain the pH value of the solution unchanged. And after the enzymolysis is finished, cooling the mixed solution to room temperature, adding 1mol/L HCl to adjust the pH value to an isoelectric point, centrifuging to collect supernatant, and freeze-drying to obtain the polypeptide.
(4) Ultrafiltration
Preparing 10mg/mL solution of pumpkin seed polypeptide, sequentially passing through 0.45-micrometer and 0.22-micrometer microporous filter membranes, adjusting ultrafiltration pressure to 0.1-0.2 MPa, and ultrafiltering at room temperature for 5 times. Collecting the polypeptide solution passing through 1000Da ultrafiltration membrane, and freeze-drying to obtain polypeptide.
(5) Sephadex G-10 isolation
Adding the pumpkin seed polypeptide with the mass concentration of 40mg/mL into the sephadex, restoring the liquid level to a position 5cm away from the gel by using ultrapure water after the sample completely permeates into the gel, and eluting by using the ultrapure water as an eluent. The elution conditions were: the elution rate was 1.0 mL/min. Collecting one tube of eluent every 5min, collecting 31-40 tubes of eluent, measuring the absorbance at 274nm, and drawing an elution curve, which is shown in figure 1 (named as F3).
FIG. 1 shows the result of Sephadex G-10 separation, which is divided into three fractions according to the peak appearance, namely F1(1-19 tubes), F2(20-30 tubes) and F3(31-40 tubes), and the peak solutions are collected together and lyophilized polypeptide powder is used to evaluate the proliferation effect of the three fractions on HSF and HaCat cells at the same mass concentration, as shown in FIG. 2, FIG. 2 a: HSF; FIG. 2 b: HaCat, component F2 has the best effect of promoting cell proliferation at the same mass concentration, so the component F2 is preferred in the invention.
Example 4
The F2 polypeptide powder prepared in example 1 was formulated into a solution and isolated using DEAE-52, specifically:
preparing 10mg/mL solution of polypeptide separated and prepared by sephadex G-10, injecting swelled DEAE-52 anion exchange resin into a chromatographic column, balancing 2 column volumes with deionized water, adding the polypeptide solution separated and prepared by sephadex G-10, and eluting samples with 0, 0.5 and 1mol/L NaCl solution at the flow rate of 1mL/min in a gradient manner respectively. 3mL of eluent is collected from each tube, and the 1 st to 50 th tube of eluent is detected at 274nm for absorbance, as shown in FIG. 3 (named F2-a).
Example 5
The F2 polypeptide powder prepared in example 1 was formulated into a solution and isolated using DEAE-52, specifically:
preparing 10mg/mL solution of polypeptide separated and prepared by sephadex G-10, injecting swelled DEAE-52 anion exchange resin into a chromatographic column, balancing 2 column volumes with deionized water, adding the polypeptide solution separated and prepared by sephadex G-10, and eluting samples with 0, 0.5 and 1mol/L NaCl solution at the flow rate of 1mL/min in a gradient manner respectively. 3mL of eluent is collected from each tube, and the 51 st to 100 th tube of eluent is detected at 274nm to obtain the absorbance shown in figure 3 (named as F2-b).
Example 6
The F2 polypeptide powder prepared in example 1 was formulated into a solution and isolated using DEAE-52, specifically:
preparing 10mg/mL solution of polypeptide separated and prepared by sephadex G-10, injecting swelled DEAE-52 anion exchange resin into a chromatographic column, balancing 2 column volumes with deionized water, adding the polypeptide solution separated and prepared by sephadex G-10, and eluting samples with 0, 0.5 and 1mol/L NaCl solution at the flow rate of 1mL/min in a gradient manner respectively. 3mL of eluent is collected from each tube, and the 101 st to 150 th tube of eluent is detected at 274nm to obtain the absorbance shown in figure 3 (named as F2-c).
FIG. 3 shows the results of DEAE-52 separation, which is divided into three fractions according to the peak appearance, namely F2-a (1-50 tubes), F2-b (51-100 tubes) and F2-c (101-150 tubes), and the peak solutions are collected together and lyophilized to obtain polypeptide powder. In subsequent experiments the proliferation effect of the three components on HSF, HaCat cells at the same mass concentration was evaluated, as shown in fig. 4, fig. 4 a: HSF, fig. 4 b: HaCat, it can be seen that component F2-b promoted cell proliferation optimally at the same mass concentration.
As shown in FIG. 11, in the HPLC chromatogram, the fraction F2-b was separated under gradient elution conditions, the peak-off time was 17.486min, and only this relatively prominent elution peak was obtained, and it can be concluded that the fraction F2-b was a single fraction. The ultraviolet absorption of the component detected by the ultraviolet detector is shown in figure 12, and the absorption peaks at 229.1nm and 272.8nm are obvious. Thus indicating that the single component F2-b is successfully separated and accords with the basic ultraviolet absorption characteristic of the polypeptide from the ultraviolet spectrum. The F2-b component low-energy TOF-MS spectrum is shown in figure 13, and the F2-b component high-energy TOF-MS spectrum is shown in figure 14.
The resulting primary mass spectra were processed using the Peptide Sequence module of the Waters Masslynx software and Peptide Sequence was deduced, the results of which are shown in figure 15. Component F2-b its amino acid sequence (SEQ ID NO. 1):
ASPNEPEDNPDGF。
for convenience in describing the invention, conventional and non-conventional abbreviations for the various amino acid residues are used. These abbreviations are familiar to those skilled in the art, but are listed below for clarity:
asp ═ D ═ aspartic acid; ala ═ a ═ alanine; arg ═ R ═ arginine;
asn ═ N ═ asparagine; gly ═ G ═ glycine; glu ═ E ═ glutamic acid;
gln ═ Q ═ glutamine; his ═ H ═ histidine; ile ═ I ═ isoleucine;
leu ═ L ═ leucine; lys ═ K ═ lysine; met ═ M ═ methionine;
phe ═ F ═ phenylalanine; pro ═ P ═ proline; ser ═ S ═ serine;
thr ═ T ═ threonine; trp ═ W ═ tryptophan; tyr ═ Y ═ tyrosine;
val ═ V ═ valine; cys ═ C ═ cysteine.
Wherein the N end of the polypeptide is alanine, and the C end is aromatic amino acid phenylalanine. The ultraviolet absorption of the polypeptide can be explained by the conjugated structure of benzene rings contained in phenylalanine residues. ASPNEPEDNPDGF was found to be a newly discovered peptide by a search query of the active peptide database (BIOPEP).
Comparative example 1
(1) Preparation of defatted pumpkin seeds
Crushing the pumpkin seeds by a high-speed crusher, and sieving by a 60-mesh sieve. Adding petroleum ether according to a feed-liquid ratio of 1:4(g/mL), stirring at 40 ℃ for 4h, performing suction filtration, repeating for 3 times, degreasing, removing residual petroleum ether in a fume hood, and storing at low temperature in a sealed manner.
(2) Preparation of pumpkin seed protein
Adding defatted semen Cucurbitae into deionized water at a ratio of 1:30g/mL, adjusting pH to 9.5, extracting at 35 deg.C for 180min, centrifuging, collecting supernatant, and adjusting pH to 4.3. Centrifuging, collecting precipitate, washing with water to neutrality, and freeze drying to obtain semen Cucurbitae protein.
(3) Preparation method of pumpkin seed polypeptide
Adjusting the temperature and pH of the pumpkin seed protein dispersion to 50 deg.C and 9.0, adding alkaline protease at an enzyme bottom ratio of 5%, performing enzymolysis for 4 hr, and inactivating in boiling water. During the enzymolysis, 0.5mol/L NaOH solution is added dropwise to maintain the pH value of the solution unchanged. And after the enzymolysis is finished, cooling the mixed solution to room temperature, adding 1mol/L HCl to adjust the pH value to an isoelectric point, centrifuging to collect supernatant, and freeze-drying to obtain the polypeptide.
(4) Ultrafiltration
Preparing 10mg/mL solution of pumpkin seed polypeptide, sequentially passing through 0.45-micrometer and 0.22-micrometer microporous filter membranes, adjusting ultrafiltration pressure to 0.1-0.2 MPa, and ultrafiltering at room temperature for 5 times. Collecting the polypeptide solution passing through 1000Da ultrafiltration membrane, and freeze-drying to obtain polypeptide.
(5) Separating and purifying
Adding the pumpkin seed polypeptide with the mass concentration of 40mg/mL into the sephadex, restoring the liquid level to a position 5cm away from the gel by using ultrapure water after the sample completely permeates into the gel, and eluting by using the ultrapure water as an eluent. The elution conditions were: the elution rate was 1.0 mL/min. Collecting one tube of eluent every 5min, collecting 20-30 tubes of eluent, and freeze-drying to obtain polypeptide;
preparing 10mg/mL solution of polypeptide separated and prepared by sephadex G-10, injecting swelled DEAE-52 anion exchange resin into a chromatographic column, balancing 2 column volumes with deionized water, adding the polypeptide solution separated and prepared by sephadex G-10, and eluting samples with 0, 0.5 and 1mol/L NaCl solution at the flow rate of 1mL/min in a gradient manner respectively. 3mL of eluent is collected for each tube, 1 st to 50 th tubes of eluent, 51 st to 100 th tubes of eluent and 101 st to 150 th tubes of eluent are collected, and the absorbance is detected at 274nm, as shown in figure 5.
As can be seen, only alkaline protease is added in the preparation process of the polypeptide, and under the condition of the same tube number, response peaks appear in 50-100 tubes, but are too small. The results show that the polypeptide prepared by the alkaline protease contains the active polypeptide combination, but the extraction efficiency is low and the yield is low. The method does not adopt an alkaline-trypsin double-enzyme composite enzymolysis mode, and has high yield.
Comparative example 2
(1) Preparation of defatted pumpkin seeds
Crushing the pumpkin seeds by a high-speed crusher, and sieving by a 60-mesh sieve. Adding petroleum ether according to a feed-liquid ratio of 1:4(g/mL), stirring at 40 ℃ for 4h, performing suction filtration, repeating for 3 times, degreasing, removing residual petroleum ether in a fume hood, and storing at low temperature in a sealed manner.
(2) Preparation of pumpkin seed protein
Adding defatted semen Cucurbitae into deionized water at a ratio of 1:30g/mL, adjusting pH to 9.5, extracting at 35 deg.C for 180min, centrifuging, collecting supernatant, and adjusting pH to 4.3. Centrifuging, collecting precipitate, washing with water to neutrality, and freeze drying to obtain semen Cucurbitae protein.
(3) Preparation method of pumpkin seed polypeptide
Adjusting the temperature and pH of the pumpkin seed protein dispersion to 50 deg.C and 9.0, adding trypsin at an enzyme bottom ratio of 5%, performing enzymolysis for 4 hr, and inactivating in boiling water. During the enzymolysis, 0.5mol/L NaOH solution is added dropwise to maintain the pH value of the solution unchanged. And after the enzymolysis is finished, cooling the mixed solution to room temperature, adding 1mol/L HCl to adjust the pH value to an isoelectric point, centrifuging to collect supernatant, and freeze-drying to obtain the polypeptide.
(4) Ultrafiltration
Preparing 10mg/mL solution of pumpkin seed polypeptide, sequentially passing through 0.45-micrometer and 0.22-micrometer microporous filter membranes, adjusting ultrafiltration pressure to 0.1-0.2 MPa, and ultrafiltering at room temperature for 5 times. Collecting the polypeptide solution passing through 1000Da ultrafiltration membrane, and freeze-drying to obtain polypeptide.
(5) Separating and purifying
Adding the pumpkin seed polypeptide with the mass concentration of 40mg/mL into the sephadex, restoring the liquid level to a position 5cm away from the gel by using ultrapure water after the sample completely permeates into the gel, and eluting by using the ultrapure water as an eluent. The elution conditions were: the elution rate was 1.0 mL/min. Collecting one tube of eluent every 5min, collecting 20-30 tubes of eluent, and freeze-drying to obtain polypeptide;
preparing 10mg/mL solution of polypeptide separated and prepared by sephadex G-10, injecting the swelled DEAE-52 anion exchange resin into a chromatographic column, balancing 2 column volumes with deionized water, adding 10mg/mL polypeptide solution separated and prepared by sephadex G-10, and eluting samples with 0, 0.5 and 1mol/L NaCl solution at the flow rate of 1mL/min in a gradient manner. 3mL of eluent is collected for each tube, 1 st to 50 th tubes of eluent, 51 st to 100 th tubes of eluent and 101 st to 150 th tubes of eluent are collected, and the absorbance is detected at 274nm, as shown in figure 6.
As can be seen, only trypsin is added in the preparation process of the polypeptide, and under the condition of the same tube number, response peaks appear in 50-100 tubes, but are too small. The results show that the polypeptide prepared by the alkaline protease contains the active polypeptide combination, but the extraction efficiency is low and the yield is low. The method does not adopt an alkaline-trypsin double-enzyme composite enzymolysis mode, and has high yield.
Comparative example 3
(1) Preparation of defatted pumpkin seeds
Crushing the pumpkin seeds by a high-speed crusher, and sieving by a 60-mesh sieve. Adding petroleum ether according to a feed-liquid ratio of 1:4(g/mL), stirring at 40 ℃ for 4h, performing suction filtration, repeating for 3 times, degreasing, removing residual petroleum ether in a fume hood, and storing at low temperature in a sealed manner.
(2) Preparation of pumpkin seed protein
Adding defatted semen Cucurbitae into deionized water at a ratio of 1:30g/mL, adjusting pH to 9.5, extracting at 35 deg.C for 180min, centrifuging, collecting supernatant, and adjusting pH to 4.3. Centrifuging, collecting precipitate, washing with water to neutrality, and freeze drying to obtain semen Cucurbitae protein.
(3) Preparation method of pumpkin seed polypeptide
Adjusting the temperature and pH of the pumpkin seed protein dispersion to 50 deg.C and 9.0, carrying out enzymolysis with protease at enzyme-substrate ratio for 4 hr, and inactivating in boiling water. During the enzymolysis, 0.5mol/L NaOH solution is added dropwise to maintain the pH value of the solution unchanged. And after the enzymolysis is finished, cooling the mixed solution to room temperature, adding 1mol/L HCl to adjust the pH value to an isoelectric point, centrifuging to collect supernatant, and freeze-drying to obtain the polypeptide.
Wherein, the protease is respectively: alkaline protease, trypsin, flavourzyme, alkaline-trypsin, alkaline-flavourzyme. In the double-enzyme enzymolysis process, the first protease is inactivated after being hydrolyzed for 2 hours, and then the second protease is used for hydrolyzing for 2 hours.
The 5 enzymolysis pumpkin seed polypeptides generated by the method are respectively named as JX, Y, F and JX-Y, JX-F. The degree of hydrolysis was measured by the pH-state method and the yield of each polypeptide was calculated. The preparation conditions of the different enzymatic products are shown in table 1.
TABLE 1
Figure BDA0002338178340000121
(4) Polypeptide yield and degree of hydrolysis
The polypeptide yield is shown in FIG. 7. As can be seen from FIG. 7, the degree of hydrolysis gradually leveled off with time, and after 4 hours the degree of hydrolysis of JX, Y, F, JX-Y, JX-F was 25.20%, 12.02%, 10.11%, 27.23%, 23.24%, respectively, with the highest degree of hydrolysis of JX-Y.
The degree of hydrolysis was measured as shown in FIG. 8, and it can be seen from FIG. 8 that the yield of JX-Y was 39.20%, which was the highest among 5. Therefore, the two indexes of the hydrolysis degree and the polypeptide yield indicate that more pumpkin seed polypeptide can be obtained by using the alkaline protease-trypsin.
(5) Molecular weight distribution
The addition of the protease enables the pumpkin seed protein chain to be cut by different degrees and different parts, and the pumpkin seed polypeptide is successfully prepared, wherein the polypeptide with the molecular weight of less than 1000Da accounts for the highest percentage of the JX-Y and reaches 92.72%. The polypeptide molecular weight distribution (%) results are shown in Table 2.
TABLE 2
Figure BDA0002338178340000131
(6) Antioxidant Properties of Polypeptides
The results of the measurement of antioxidant properties of the polypeptides are shown in FIG. 9. As can be seen from FIG. 9, the oxidation resistance is sequentially arranged from small to large: y is more than F and less than JX and JX-F is more than JX-Y, wherein JX-Y has the optimal oxidation resistance. When the concentration is 10mg/mL, the clearance rates of the free radicals to DPPH, OH and O2 are 46.27%, 25.37% and 10.57%, respectively.
(7) Promoting cell proliferation
HSF and HaCat are important components of human skin cells, wherein HSF is located in the dermis layer of the skin, and HaCat is located in the epidermis layer. The effect of the polypeptide on cell proliferation is shown in fig. 10, fig. 10 a: HSF; FIG. 10 b: the JX-Y in the HaCat and 5 pumpkin seed polypeptides has the optimal performance of promoting cell proliferation, and the activities of the HSF and HaCat cells respectively reach 116.15% and 121.02%.
The invention provides a preparation method of polypeptide capable of promoting human skin cell proliferation, wherein a polypeptide compound is extracted from pumpkin seeds for the first time, the extracted polypeptide compound can obviously promote the proliferation of human skin fibroblasts and human immortalized keratinocytes, can be used as a repairing agent and an anti-aging component to be added into skin care products, and has wide market prospect.
The inventor finds that in the preparation process of the pumpkin seed polypeptide, the desalted pumpkin seed protein is used as a raw material, and the pumpkin seed polypeptide is prepared by preferably performing composite enzymolysis on alkaline-trypsin double enzymes, so that the yield of the polypeptide is higher, the molecular weight of the polypeptide is less than 1000Da and reaches 92.72%, the antioxidation performance is optimal, the cell proliferation promoting performance is better, and the activities of HSF (high-speed lipoprotein lipase) and HaCat (Hazarin lipase) cells respectively reach 116.15% and 121.02%.
Preparing 1-50 mg/mL solution of pumpkin seed polypeptide, adjusting pH to 5.0-10.0, centrifuging, collecting supernatant, sequentially passing through 0.45-micron and 0.22-micron microporous filter membranes, adjusting ultrafiltration pressure to 0.1-0.2 MPa, carrying out ultrafiltration for 1-10 times at normal temperature, collecting polypeptide solution passing through a 1000Da ultrafiltration membrane, and freeze-drying to obtain polypeptide powder; adding the pumpkin seed polypeptide prepared by ultrafiltration into sephadex G-10, eluting with ultrapure water as an eluent after the sample completely permeates into the sephadex, collecting the eluent, and freeze-drying to obtain polypeptide powder; preparing 1-50 mg/mL solution of polypeptide separated and prepared by sephadex G-10, adjusting pH to 5.0-10.0, centrifuging, collecting supernatant, and sequentially passing through 0.45-micrometer and 0.22-micrometer microporous filter membranes to obtain screened pumpkin seed polypeptide; injecting the swelled DEAE-52 anion exchange resin into a chromatographic column, balancing 2 column volumes by deionized water, adding the screened pumpkin seed polypeptide, performing gradient elution on the sample by using 0, 0.5 and 1mol/L NaCl solutions at the flow rate of 1mL/min respectively, eluting 3mL of the eluent in each tube, preferably collecting the eluent in the 50 th to 100 th tubes, and performing freeze drying to obtain the polypeptide capable of promoting human skin cell proliferation, wherein the polypeptide has the optimal cell proliferation promoting performance.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Sequence listing
<110> university of south of the Yangtze river
<120> preparation method and product of polypeptide capable of promoting human skin cell proliferation and application thereof
<141> 2019-12-18
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 13
<212> PRT
<213> Cucurbita moschata
<400> 1
Ala Ser Pro Asn Glu Pro Glu Asp Asn Pro Asp Gly Phe
1 5 10

Claims (7)

1. A method for preparing polypeptide capable of promoting human skin cell proliferation is characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
preparing 1-50 mg/mL solution of pumpkin seed polypeptide, adjusting pH to 5.0-10.0, centrifuging, collecting supernatant, sequentially passing through 0.45-0.22-micrometer microporous filter membranes, adjusting ultrafiltration pressure to 0.1-0.2 MPa, carrying out ultrafiltration for 1-10 times at normal temperature, collecting polypeptide solution passing through a 1000Da ultrafiltration membrane, and freeze-drying to obtain polypeptide powder;
adding the pumpkin seed polypeptide prepared by ultrafiltration into sephadex G-10, eluting by using ultrapure water as an eluent after a sample completely permeates into the sephadex, collecting the eluent of a 20 th-30 th tube, and freeze-drying to obtain polypeptide powder; wherein the elution condition is that the elution speed is 1.0mL/min, and one tube of eluent is collected every 5 min;
preparing 1-50 mg/mL solution of polypeptide separated and prepared by sephadex G-10, adjusting pH to 5.0-10.0, centrifuging, collecting supernatant, and sequentially passing through 0.45-micrometer and 0.22-micrometer microporous filter membranes to obtain screened pumpkin seed polypeptide;
injecting the swelled DEAE-52 anion exchange resin into a chromatographic column, balancing 2 column volumes with deionized water, adding the screened pumpkin seed polypeptide, performing gradient elution on the sample by using 0, 0.5 and 1mol/L NaCl solutions at the flow rate of 1mL/min respectively, eluting 3mL in each tube, collecting the eluates of 50 th to 100 th tubes, and performing freeze drying to obtain the polypeptide capable of promoting human skin cell proliferation; wherein the content of the first and second substances,
the preparation method of the pumpkin seed polypeptide comprises the following steps: adding desalted pumpkin seed protein into deionized water according to a material-liquid ratio of 1: 5-1: 40g/mL, performing ultrasonic treatment for 10-60 min at a power of 20-60 Hz to obtain a pumpkin seed protein dispersion liquid, adjusting the temperature to 30-60 ℃ and the pH to 5.0-10.0, adding alkaline protease according to an enzyme bottom ratio of 1% -5.5%, performing enzymolysis for 2-4 h, then performing inactivation, adjusting the temperature of a reaction liquid to 25-50 ℃, the pH to 7.5, adding trypsin according to an enzyme bottom ratio of 1% -5.5%, performing enzymolysis for 2-4 h, then performing inactivation, cooling the mixed liquid to room temperature, adding 1mol/L HCl to adjust the pH to 7.0, centrifuging for 25min at 5000r/min to collect supernatant, and performing freeze drying to obtain the pumpkin seed polypeptide.
2. The method for preparing polypeptide capable of promoting human skin cell proliferation according to claim 1, wherein: and (3) performing freeze drying to obtain the polypeptide capable of promoting human skin cell proliferation, wherein the temperature of a cold trap is-50 to-45 ℃, the vacuum degree is 0.025MPa, and the drying time is 24 h.
3. The method for preparing polypeptide capable of promoting human skin cell proliferation according to claim 1, wherein: preparing 1-50 mg/mL solution of pumpkin seed polypeptide, adjusting the pH value to 5.0-10.0, centrifuging and collecting supernatant, wherein the centrifugation condition is 8000r/min, and the time is 10 min.
4. The method for preparing polypeptide capable of promoting human skin cell proliferation according to claim 1, wherein: the desalted pumpkin seed protein and the preparation method thereof comprise,
adding degreased pumpkin seeds into deionized water according to a material-liquid ratio of 1: 10-1: 50g/mL, adjusting the pH value to 7.0-11, performing ultrasonic extraction at 25-50 ℃ for 240min, wherein the ultrasonic power is 20Hz in the first 120min, the ultrasonic power is 40Hz in the second 120min, centrifuging, collecting supernatant, adjusting the pH value to 7.0, centrifuging at 5000r/min for 25min, collecting precipitate, washing with water to remove salt, and freeze-drying to obtain desalted pumpkin seed protein.
5. The method for preparing polypeptide capable of promoting human skin cell proliferation according to claim 4, wherein: the preparation method of the degreased pumpkin seeds comprises the following steps,
crushing the pumpkin seeds by a high-speed crusher, sieving by a 100-mesh sieve, adding petroleum ether according to the material-liquid ratio of 1: 2-1: 5g/mL, stirring for 4 hours at 25-50 ℃, performing suction filtration, repeating for three times, degreasing, removing residual petroleum ether in a fume hood, and storing at low temperature in a sealed manner.
6. A polypeptide prepared by the preparation method of any one of claims 1 to 5, wherein: the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.
7. Use of the polypeptide according to claim 6 as an anti-aging, repairing, anti-oxidant ingredient in cosmetics.
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